Improved flow cytometric detection of HLA alloantibodies using pronase - Potential implications in renal transplantation

S. Vaidya et al., Improved flow cytometric detection of HLA alloantibodies using pronase - Potential implications in renal transplantation, TRANSPLANT, 71(3), 2001, pp. 422-428
Citations number
Categorie Soggetti
Medical Research Diagnosis & Treatment
Journal title
ISSN journal
0041-1337 → ACNP
Year of publication
422 - 428
SICI code
Background Flow cytomeric crossmatch (FCXM) has grown in popularity and has become the "standard of practice" in many programs. Although FCXM is the m ost sensitive method for detecting alloantibody, the B cell FCXM has been p roblematic. Difficulties with the B cell FCXMs have been centered around hi gh nonspecific fluorescence background owing to Fc-receptors present on the B cells and autoantibodies, To improve the specificity and sensitivity of the B cell FCXM, we utilized the proteolytic enzyme pronase to remove Fc re ceptors from lymphocytes before their use in FCXM. Methods. Lymphocytes isolated from peripheral blood, spleen, or lymph nodes were treated with pronase and then used in a three-color FCXM, A total of 167 T- and B cell FCXMs using pronase-treated and untreated cells were perf ormed, Testing used serial dilutions of HLA allosera (22 class I and 6 clas s PI), with the titer of each antibody at one dilution past the titer at wh ich the complement-mediated cytotoxicity anti-human globulin crossmatch bec ame negative. Results. After pronase treatment, the actual channel values of the negative control in both T cell and B cell FCXMs declined from 78+/-10 to 57+/-4 (P <0.05) and 107+/-11 to 49+/-3 (P<0.00001), respectively. Pronase treatment resulted in improved sensitivity of the T and B cell FCXM in detecting clas s I antibody by 20% and 80%, respectively, In no instance was a false-posit ive reaction observed. In this study, pronase treatment improved the specif icity of B cell FCXM for detecting class II antibodies from 75% to 100% (P= 0.03), In no instance was a false-negative reaction recorded. Lastly, on th e basis of these observations me re-evaluated three primary transplant reci pients who lost their allografts because of accelerated rejection. One of t he patients was transplanted across negative T and B cell FCXM, whereas the other two patients were transplanted across a positive T cell, but negativ e B cell, FCXM. After pronase treatment, T and B cell FCXMs of each patient became strongly positive, and donor-specific anti-HLA class I antibody was identified in each case. Conclusion, Utilization of pronase-treated lymphocytes improves both the se nsitivity and specificity of the FCXM.